Enzymatic process for preparing a synthetic ester from a vegetable oil

ABSTRACT

PCT No. PCT/FI95/00478 Sec. 371 Date Mar. 4, 1997 Sec. 102(e) Date Mar. 4, 1997 PCT Filed Sep. 7, 1995 PCT Pub. No. WO96/07751 PCT Pub. Date Mar. 14, 1996A two step process for preparing a synthetic ester from a vegetable oil by means of lipase enzymes. A lubricant composition comprising a synthetic ester prepare by said process.

The objects of the present invention are a process for preparing asynthetic ester from a vegetable oil by means of lipase enzymes, andlubricants which contain a synthetic ester prepared by said process.

Natural fats and oils have been used as lubricants already for thousandsof years. With industrialization mineral based lubricants came also tothe market. The applications of lubricants and thus also therequirements set for them have changed and developed with the advance oftechnology. Various types of synthetic esters and lubricants containingthe same have been developed to meet the new requirements.

The purpose of a lubricant is to minimize friction and wear of metals.Lubricants are developed according to the use and they consist of a basefluid and additives improving the lubricative properties. With thedevelopment of technology, lubricants are used under more and moresevere conditions, such as at very low or very high temperatures (e.g.the turbine engines of aeroplanes). At the same time biodegradability,non-accumulation to the environment, non-toxicity and the use ofrenewable raw materials have emerged as new requirements. The use ofbiodegradable lubricants is of particular importance in the machines anddevices used in the fields of agriculture, forestry and building, as theoil used may be left in the environment.

By the synthetic esters developed as lubricants are meant estersprepared from mono-, di- or trialcohols and mono- or dicarboxylic acidsby known esterification and transesterification methods. Theconventional chemical process comprises combining all the reactants andletting them react in one stage. The reaction may be carried out in thepresence of catalysts, such as acids, bases or metal oxides. In additionto chemical agents, also lipase enzymes can act as catalysts oftransesterification reactions.

Lipases (triacylglycerol acylhydrolase; EC 3.1.1.3) belong to theesterase enzyme group, and fats and oils are their natural substrates.Several microbes (yeasts, molds, bacteria) secrete in their growth medialipases by means of which lipids decompose into nutrients of themicrobe. Lipases catalyze the hydrolysis reactions of oils and fats butunder suitable conditions they also catalyze the synthesis andtransesterification of tri-, di- and monoglyceride esters (Yamane etal., J. Am. Oil Chem. Soc. 64, 1987, 1657-1662).

On the basis of their specificity, lipases are divided into threegroups, nonspecific, 1,3-specific and fatty acid specific lipases.Nonspecific lipases are produced by for instance the yeast Candidarugosa (ex. cylindracae) and the bacteria Corynebacterium acnes andStaphylococcus aureus. Nonspecific lipases release fatty acids from allthree positions of a triglyceride. According to their name, 1,3-lipasesrelease fatty acids from positions 1 and 3 of triglycerides. Theselipases are produced by for instance the molds Aspergillus niger, Mucorjavanicus, Mucor miehei and Rhizopus arrhizus as well as by the yeastCandida lipolytica. The fatty acid specific lipases release only certainfatty acids from triglycerides. Mucor miehei, for example, produces alsoa lipase which in addition to 1,3-specificity is also specific to fattyacids with 12 carbon atoms. However, the specificity is not absolute.

The structure of the synthetic ester used has a profound effect on thestability of the lubricant. Esters decompose by the effect of heatand/or oxygen. It is known to increase the thermal stability ofsynthetic esters by using in the preparation no beta hydrogen alcohols.Oxidative properties on the other hand can be improved by deuteration ofesters.

Synthetic esters intended for a lubricative use are classified bystructure as monocarboxylic acid, dicarboxylic acid, polyol and complexesters. Due to their low viscosity and high volatility monoesters arepoorly suitable as lubricants. Polyol esters are chemically more stablethan for example diesters, due to the structure of the polyols used inthe preparation of said esters wherein no hydrogen atom is attached tothe β carbon atom. Complex esters have promising lubricative propertiesbut the manufacture thereof on an industrial scale is difficult becauseof the severe conditions required by the reaction, especially if saidesters are prepared from (purified) fatty acids and alcohols.

If polyol esters are prepared by using no alfa hydrogen acids, thestability properties of the esters can be further improved. Metro et al.(CA 859 771) have shown that the no alfa hydrogen carboxylic acidsincrease the thermal and oxidative stability of esters prepared from nobeta hydrogen alcohols, as well as slow down the hydrolysis of theesters.

As the low viscosity polyol esters are not suitable for traditional useswherein high viscosity is required, it has been aimed at preparingpolyol esters of higher viscosity from for example trimethylol propane(TMP). However, it has been found that it is difficult to obtain simpleTMP esters with both high viscosity and a low pour point (cf. forexample U.S. Pat. No. 4,061,581).

Products based on vegetable oils are nowadays used more and more aslubricants because of their safety to the environment. Natural vegetableand animal oils are glyceride diesters, i.e. tri-, di- or monoesters ofglycerol and straight chain saturated and unsaturated fatty acids. Thelubricant industry uses for instance rapeseed, rape, soybean, castor,olive, coconut, palm and tall oils.

The advantageous properties of vegetable oils include user friendlinessand non-toxicity. In addition, vegetable oils are renewable rawmaterials and degrade in the environment without accumulating in thefood chain of nature. However, the use of vegetable oils as lubricantshas been limited by their poor stability properties. The poor thermaland oxidative stability is due to unsaturated and polyunsaturated fattyacids. On the other hand, the unsatisfactory behaviour of vegetable oilsat low temperatures is due to the saturated fraction of fatty acids. Byusing suitable additives and by favouring in cultivation such varietieswhich do not have a too high degree of saturation, it has been possibleto somewhat improve the stability properties. Also the purification ofthe oil for technical use is helpful.

Furthermore, attempts have been made to modify natural glyceride estersin order to improve their stability properties. Known processes includecatalytic hydrogenation, alcoholysis, geometrical isomerization andsulfurization. For example by hydrogenation a certain amount of doublebonds from the unsaturated part of vegetable oils can be removed, and byisomerization the amount of undesired isomers can be decreased.

Van der Waal and Kenbeek have presented a process for the preparation ofsynthetic esters from vegetable oils or animal fats (Proceedings of theTribology 2000, 8th International Colloqium, Technische AkademieEsslingen, Germany, 14-16 June 1992, Vol II, pp 13.3-1-13.3-8). Theprocess comprises first decomposing the glyceride esters of the startingmaterial into fatty acids and glycerol and subsequently separating thefatty acid fraction into liquid and solid phases. The fatty acids of theliquid phase are separated by distillation into single fatty acids whichcan be further modified e.g. by hydrogenation or cracking to obtain thedesired raw material. Fractions containing a single fatty acid areesterified with no beta hydrogen polyols for preparing a syntheticester.

The fatty acids of the ester prepared according to the above describedprocess usually contain less unsaturated double bonds than the fattyacids of the starting material, which improves the oxidative stability.However, the costs of the process are extremely high, due to themultistage separation and purification reactions and the most severeconditions (high pressure and temperature) required by the reaction.Moreover, it has been found that when fractions containing only a singlefatty acid are reacted with polyols, plenty of mono- and diglyceridesare formed, i.e. all the OH groups of the polyols do not react. Thisdecreases the triglyceride yield and the raw material has to be recycledseveral times if the yield is to be improved. Furthermore, the reactionof a fatty acid and an alcohol creates water which has to be removedduring the reaction.

Transesterification of fats by means of lipases is known as such. Theliterature in the field discloses especially various systems for theimmobilization of the lipases used (cf. for example EP patentapplication 579 928 and U.S. Pat. Nos. 4,798,793 and 4,818,695). Theimmobilization of lipases facilitates their application both incontinuous and batch processes. Patent publication GB 1 577 933discloses a process for modifying triglycerides with a lipase,especially with an immobilized lipase. However, the literature in theart does not describe the use of lipases as a catalyst in the processaccording to the present invention.

According to the invention it has now been found that it is possible toprepare synthetic esters with good lubricative properties from vegetableoils by an enzymatic process which avoids the multistage reaction withseveral separations and recyclings and by which good yields areobtained.

In the process according to the invention a vegetable oil is firsttransesterified by reacting the vegetable oil with a lower alkanol toobtain a mixture of fatty acid lower alkyl esters. The process ischaracterised in that the mixture of esters obtained from the firstreaction is further transesterified by reacting said mixture with a nobeta hydrogen polyol of the formula ##STR1## wherein R is a C₁ -C₆ alkylgroup, particularly a C₁ -C₄ alkyl group, or a --CH₂ OH group, in thepresence of a lipase enzyme, and the synthetic ester obtained isrecovered.

Vegetable oils suitable as a starting material in the process are forexample rapeseed, rape, soybean, castor, olive, coconut, palm, tall,maize, walnut, flaxseed, cotton, sunflower, sesame and almond oils,especially rapeseed oil, rape oil, tall oil and soybean oil,particularly rapeseed oil or rape oil.

The first transesterification reaction of the process according to theinvention is carried out by a process known per se, by reacting arefined or alkalirefined vegetable oil with a lower alkanol to obtain amixture of fatty acid lower alkyl esters.

The lower alkanol used in the first transesterification reaction ispreferably a C₁ -C₄ alkanol, especially methanol or ethanol. Theobtained mixture of lower alkyl esters of the vegetable oil is thuspreferably a mixture of C₁ -C₄ alkyl esters, especially a mixture ofmethyl or ethyl esters. If desired, usual esterification catalysts maybe used in the reaction, and the amounts of the reactants and thereaction conditions (pressure, temperature, reaction time) are eithercommonly known or easily chosen by a person skilled in the art. Thereaction may also be carried out by using a suitable enzyme as acatalyst.

The first transesterification reaction may be illustrated by thefollowing general reaction scheme I: ##STR2## wherein R₁, R₂ and R₃ arefatty acid residues, R₄ is an alkyl residue, especially a C₁ -C₄ alkylresidue, and R_(x) is R₁, R₂ or R₃. Glycerol is formed as a by-product.

The fatty acid lower alkyl ester obtained from the firsttransesterification reaction is thus a mixture comprising various fattyacids of the vegetable oil used as the starting material. It is typicalof the invention that this mixture of fatty acid lower alkyl esters maybe used directly as the starting material of the secondtransesterification reaction without separation or purification of fattyacids.

In the second transesterification reaction according to the invention,the mixture of fatty acid lower alkyl esters obtained from the firsttransesterification reaction is reacted with a no beta hydrogen polyol,such as for example trimethylol ethane, trimethylol propane, trimethylolbutane or pentaerythritol, especially with pentaerythritol ortrimethylol propane, in the presence of a lipase.

The second transesterification reaction may be illustrated with thefollowing general reaction scheme II: ##STR3## wherein R₄ and R_(x) havethe same meanings as in the reaction scheme I and R is a C₁ -C₆ alkylgroup, especially a C₁ -C₄ alkyl group, or a --CH₂ OH group.

Consequently, it is the question of a totally different chemicalreaction than in the process of the prior art wherein a free fatty acidis esterified with an alcohol. In the process according to theinvention, an ester is reacted with an alcohol, and thus it is thequestion of a transesterification reaction which reaction, as well asthe reaction conditions required by it and the by-products formedtherein, is totally different from the reaction used in the process ofthe prior art.

The synthetic ester obtained from the second transesterificationreaction is recovered and, if desired, purified by conventional methods,for example by neutralization and washing with an aqueous acid. Nodistillation or any other special treatment is needed as the obtainedester is ready to use as such as a raw material of lubricants.

When a polyol is reacted with a mixture of fatty acid lower alkyl estersin the presence of a suitable lipase, almost all OH-groups of the polyolreact into triglycerides. From 75 to 98% of the theoretical yield of thetriglyceride is obtained, the proportion of mono- and diglycerides beingin total from about 2 to 25%. The product obtained does not contain anyfree fatty acids which makes it an especially advantageous raw materialfor lubricants wherein the oxygenation of free fatty acids would causeproblems (corrosion, change of viscosity). The process is well adaptedfor industrial scale and the synthetic ester obtained has betterstability properties than the vegetable oil used as the raw material,while at the same time the advantageous properties of a vegetable oil(biodegradability, non-toxicity, user friendliness) are maintained.

By the process according to the invention it is thus possible to preparesynthetic esters from vegetable oils, for example from rapeseed oil, ina yield of even over 95% of the theoretical. In this case, the di- andmonoglycerides of the product are also calculated in the yield. Duringthe tests carried out it has been observed that the advantageousproperties of the product are maintained in spite of the moderate (up to30%) di- and monoglyceride content.

The no beta hydrogen polyol and the mixture of esters are preferablyreacted with each other in a molar ratio of about 1:2 to 1:6, especiallyin the molar ratio of about 1:3 to 1:3,5.

The second transesterification reaction, being characteristic of theinvention, is preferably carried out in a reduced pressure generatorprovided with reflux, for example under negative pressure of 2.0 to 12MPa, preferably under negative pressure of 5.3 MPa. The reaction iscarried out at a temperature wherein the lipase used is active, forexample at a temperature between 37° C. and 69° C., preferably at atemperature between 42° C. and 47° C. A suitable reaction time is from24 hours up to 72 hours, depending on the other conditions and theenzyme used. It is preferred to add water to the reaction mixture, forexample about 0.1-29%, preferably 8-15%, or to carry out at a highertemperature without adding water. The amount of the enzyme is preferablyfrom about 2% up to about 50% calculated (w/w) on the substrates. With a68 hour reaction, a methyl ester of rapeseed oil is completely made toreact into products only with an enzyme amount of 10%. The amount of theenzyme needed may be decreased by immobilizing the enzyme. In theprocess according to the invention, a lipase obtained for example fromCandida rugosa (ex. cylindraceae), Mucor miehei or Pseudomonasfluorescens may be used. The lipase may also be produced by transforminga gene coding for the desired enzyme into another host organism, bycultivating the host thus obtained and by isolating the lipase producedby it. Commercially obtainable immobilized lipases may be used, or thefree lipase may be immobilized before use for example on an ion exchangeresin, adsorption resin, celites, diatomaceous earth or silica gelaccording to the conventional immobilization methods.

The synthetic ester prepared by the process according to the inventionis an excellent raw material for the preparation of lubricants.Lubricants, especially hydraulic oils, which contain a synthetic esterprepared by the process of the invention, optionally with one moreadditives, are also included in the scope of the invention. As additivesfor example oxidation inhibitors, antiwear agents, antifoam agents,corrosion inhibitors, dispersants, viscosity index improvers and/or pourpoint depressers which are generally known in the art, may be used.

Oxidation inhibitors include for example amines and phenols. As antiwearagents and corrosion inhibitors for example phosphates or sulfonates andas antifoam agents for example metal sulfonates, metal phenates,polyesters or silicones may be used. Viscosity index improvers includefor example polyisobutenes, styrene-butadiene andethene-propene-copolymers which all are thus suitable also as pour pointdepressors.

In the following the invention is further described by means ofexamples, the purpose of which is to illustrate but not to limit theinvention.

EXAMPLE 1

A methyl ester of rapeseed oil was prepared as follows: Rapeseed oil(0.3 moles) was weighed into a three-necked flask provided with athermometer, cooler and a stirring device. Stirring was started andmethanol (2.0 moles) was added. The reaction mixture was heated to 60°C. and the alkali catalyst used was added (0.5%, w/w). Stirring wascontinued for three hours. The progress of the reaction was followed bythin layer chromatography. The reaction mixture was washed with anaqueous acid. The glycerol created in the reaction mixture was separatedand the product mixture was analyzed. Rapeseed oil ester content was97%.

EXAMPLE 2

In a 25 cm³ round bottom flask attached to a Liebig-refluxer of 20 cmwith a cold (about +6° C.) tap water circulating in the cooling jacket,was weighed 0.607 g (4.52 mmoles) of solid trimethylol propane (Merck,Darmstadt, Germany), and 0.7 ml of destined water was added. Afterdissolution, 4.00 g (13.56 mmoles) of methylated rapeseed oil (RaisionYhtyma, Finland) was added and finally 1.79 g of Candida rugosa lipase(Biocatalysts Ltd., Pontypridd, Great Britain) in powder form. Anegative pressure of 5.3 MPa was sucked into the device. For stirring amagnetic stirrer was attached to the device. The reaction mixture wasstirred with the magnetic stirrer at a speed of 200 rpm. The startingpoint of the reaction was counted from the moment the suction for thereduced pressure was connected to the device. The reaction temperaturewas 42° C. and the total reaction time 72 hours. The amount ofsubstituted TMP esters in the final product was over 98% in total.

EXAMPLE 3

Example 2 was repeated with 1.84 g of a Mucor miehei lipase Lipozyme IM(Novo Nordisk A/S, Bagsvaerd, Denmark) bound to a solid support. Waterwas not added to the reaction mixture. The reaction temperature was 58°C. The TMPE content of the final product was 75.0% after 24 hours and92.5% after 66 hours. There were no starting materials left after 66hours.

EXAMPLE 4

Example 2 was repeated with 1.84 g of a Candida rugosa lipase bound to asolid support. 0.7 ml water was added to the reaction mixture. Thereaction temperature was 47° C. The TMPE content of the final productwas 62.7% after 48 hours and 72.9% after 78 hours.

The enzyme bound to the solid support was prepared as follows: 3.33 g oflipase was dissolved in 100 ml of 0.05M sodium phosphate buffer, stirredfor 2 hours and filtrated. To an erlenmeyer flask of 250 ml 40 g of abuffered support (e.g. MWA-1, Mitsubishi Chemical Company, Japan; 43.4%dry matter) and 60 ml of enzyme solution (2 g lipase) was added, shakenfor 3 hours at a speed of 130 rpm, filtrated and lyophilized for 30hours to a dry solids content of 98.9%

EXAMPLE 5

Preparation of a Hydraulic Oil from a Rapeseed Oil Ester and Comparisonof Hydraulic Oils

The raw material used was the synthetic rapeseed oil ester obtained inExample 2. Said ester was mixed at a certain temperature with additivesto obtain a hydraulic oil having the following composition:

    ______________________________________                                        The synthetic ester from Example 2                                                                 90-98% by weight                                         Oxidation inhibitor  0.1-2.5% by weight                                       Pour point depresser 0-5.0% by weight                                         Antiwear agent       0.1-2.0% by weight                                       Antifoam agent       0-0.5% by weight                                         ______________________________________                                    

The technical properties studied of this ester containing additives werewearing, friction, oxidation, low temperature properties and corrosion.

Wear and friction were examined with a four ball test (ASTMD 2783, IP239) wherein wear with respect to loading or the extreme loading wherethe lubrication still works, are measured. Oxidative properties werestudied with an oxygen bomb test (ASTMD 925) and with the oxidation testDIN 51586 where the change of viscosity at 40° C. was monitored. In acorrosion test (Cincinnati-Milacron test) the aging of the oil as wellas copper and steel corrosion were studied. In said test, the change ofthe total acid number (TAN) and viscosity, the weight change of thecopper and steel rods used as oxidation catalysts in the test procedureand the formation of a precipitate under the test conditions aremeasured. Furthermore, the pour point which illustrates the lowtemperature properties of an oil was analyzed, i.e. the temperaturewhere the oil is still fluid.

The corresponding properties were examined also from hydraulic oils ofthe state of the art containing the same additives and from hydraulicoils based directly on rapeseed oil containing also the same additives.The results are shown in Table 1.

                  TABLE 1                                                         ______________________________________                                        Comparison of the properties of hydraulic oils. A = hydraulic oil with        the ester prepared by the process of the invention as raw material,           viscosity grade 32; B1 and B2 = hydraulic oils with commercial                synthetic esters as raw materials, viscosity grades 46 and 68;                C = commercial hydraulic oil based on rapeseed oil,                           viscosity grade 32.                                                                          A     B1      B2      C                                        ______________________________________                                        Four ball test                                                                extreme loading, N                                                                             2500    3000    2500  2000                                   wearing, mm      0.42    0.46    0.41  0.64                                   Oxygen bomb test                                                              ASTDM D445, psi  40      39      29    30                                     Oxidation inhibition test                                                     DIN 51586, viscosity change,                                                                   11.5    20.3    24.1  28.8                                   Cincinnati-Milacron test                                                      TAN mg KOH/g                                                                  before           1.38    1.39    1.40  1.72                                   after            1.58    3.71    2.41  0.61                                   TAN              0.20    2.32    1.01  1.11                                   viscosity change, %                                                                            19.0    16.9    6.2   8.2                                    total precipitate, mg/100 ml                                                                   1.0     17.0    28.8  4.4                                    weight change of Cu rod, mg                                                                    1.5     -16.9   0     -0.5                                   weight change of steel rod,                                                                    0.2     0.4     1.2   -0.5                                   mg                                                                            Pour point, °C.                                                                         -41     -36     -39   -39                                    ______________________________________                                    

From the results it can be seen that as regards low temperatureproperties, the ester prepared by the process according to the inventionis equal to the commercial raw materials on the market and better thanthe commercial product based on rapeseed oil. From theCincinnati-Milacron test it can be seen that the change of the totalacid number (TAN) is clearly the lowest with the ester of the invention.The increase in viscosity at 40° C. is of the same order with all, aswell as the weight change of copper and steel rods. The results of theoxygen bomb test are equal, as well as the results of the test accordingto DIN 51586 and the four ball test.

We claim:
 1. An enzymatic process for preparing a synthetic ester from avegetable oil, comprisingtransesterification of said vegetable oil byreacting it with a lower alkanol to form a mixture of lower alkyl estersof fatty acids, a second transesterification reaction wherein theobtained mixture of esters is reacted in the presence of a lipasetriacylglycerol acylhydrolase; EC 3.1.1.3, with a no beta hydrogenpolyol of the formula ##STR4## wherein R is a C₁ -C₆ alkyl group or a--CH₂ OH group, and recovering the synthetic ester obtained.
 2. Theprocess according to claim 1, wherein the vegetable oil is rapeseed oil.3. The process according to claim 1, wherein the lower alkanol is a C₁-C₄ alkanol.
 4. The process according to claim 1, wherein the fatty acidlower alkyl ester is a methyl ester of a fatty acid.
 5. The processaccording to claim 1, wherein the no beta hydrogen polyol is selectedfrom the group consisting of trimethylol ethane, trimethylol propane,trimethylol butane and pentaerythritol.
 6. The process according toclaim 1, wherein the second transesterification reaction is carried outin the presence of an immobilized lipase.
 7. The process according toclaim 1, wherein the second transesterification reaction is carried outin the presence of a Candida rugosa lipase.
 8. The process according toclaim 1, wherein the second transesterification reaction is carried outin the presence of a Mucor miehei lipase.
 9. The process according toclaim 1, wherein the lipase is separated after the reaction andrecycled.
 10. The process according to claim 1, wherein the secondtransesterification reaction is carried out with a lipase obtained bytransforming a gene coding for said enzyme into another host organismfor producing the lipase.
 11. The process according to claim 1, whereinthe reaction mixture contains about 0.1 to 29% water.
 12. The processaccording to claim 1, wherein the reaction temperature in the secondtransesterification is between 37° C. and 69° C.
 13. The processaccording to claim 1, wherein the no beta hydrogen polyol and themixture of esters are reacted with each other in a molar ratio of fromabout 1:2 to 1:6.
 14. A lubricant composition comprising a syntheticester obtained according to claim 1, optionally with one or moreadditives.
 15. The lubricant composition according to claim 14 whichcomprises about 90 to 98% of a synthetic ester and about 2 to 10% ofadditives.
 16. The lubricant composition according to claim 14 whereinthe additive is selected from the group consisting of an oxidationinhibitor, an antiwear agent, an antifoam agent, a corrosion inhibitor,a dispersant, a viscosity index improver, a pour point depresser andmixtures thereof.
 17. The process according to claim 2, wherein thesecond transesterification reaction is carried out in the presence of animmobilized lipase.
 18. The process according to claim 3, wherein thesecond transesterification reaction is carried out in the presence of animmobilized lipase.
 19. The process according to claim 4, wherein thesecond transesterification reaction is carried out in the presence of animmobilized lipase.
 20. The process according to claim 1, wherein thelower alkanol is methanol or ethanol.